Search results for: cellulosome

Authors: C. C. Lin, S. C. Kan, C. W. Yeh, C. I Chen, C. J. Shieh, Y. C. Liu

Abstract:

In this study, lipid-deprived residuals of microalgae were hydrolyzed for the production of reducing sugars by using the recombinant Bacillus cellulosome, carrying eight genes from the Clostridium thermocellum ATCC27405. The obtained cellulosome was found to exist mostly in the broth supernatant with a cellulosome activity of 2.4 U/mL. Furthermore, the Michaelis-Menten constant (Km) and Vmax of cellulosome were found to be 14.832 g/L and 3.522 U/mL. The activation energy of the cellulosome to hydrolyze microalgae LDRs was calculated as 32.804 kJ/mol.

Keywords: lipid-deprived residuals of microalgae, cellulosome, cellulose, reducing sugars, kinetics

Procedia PDF Downloads 363

Authors: Jessica Pinheiro Silva, Brenda Rabello De Camargo, Daniel Gusmao De Morais, Eliane Ferreira Noronha

Abstract:

The utilization of lignocellulosic biomass as source of polysaccharides for industrial applications requires an arsenal of enzymes with different mode of action able to hydrolyze its complex and recalcitrant structure. Clostridium thermocellum is gram-positive, thermophilic bacterium producing lignocellulosic hydrolyzing enzymes in the form of multi-enzyme complex, termed celulossomes. This complex has several hydrolytic enzymes attached to a large and enzymically inactive protein known as Cellulosome-integrating protein (CipA), which serves as a scaffolding protein for the complex produced. This attachment occurs through specific interactions between cohesin modules of CipA and dockerin modules in enzymes. The present work aims to construct celulosomes in vitro with the structural protein CipA, a xylanase called Xyn10D and a cellulose called CelJ from C.thermocellum. A mini-scafoldin was constructed from modules derived from CipA containing two cohesion modules. This was cloned and expressed in Escherichia coli. The other two genes were cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9 and integrated into the genome of the methylotrophic yeast Pichia pastoris GS115. Purification of each protein is being carried out. Further studies regarding enzymatic activity of the cellulosome is going to be evaluated. The cellulosome built in vitro and composed of mini-CipA, CelJ and Xyn10D, can be very interesting for application in industrial processes involving the degradation of plant biomass.

Keywords: cellulosome, CipA, Clostridium thermocellum, cohesin, dockerin, yeast

Procedia PDF Downloads 202

Authors: Nisha Singh, Munish Puri, Collin Barrow, Deepak Tuli, Anshu S. Mathur

Abstract:

The biological conversion of lignocellulosic biomass to ethanol is a promising strategy to solve the present global crisis of exhausting fossil fuels. The existing bioethanol production technologies have cost constraints due to the involvement of mandate pretreatment and extensive enzyme production steps. A unique process configuration known as consolidated bioprocessing (CBP) is believed to be a potential cost-effective process due to its efficient integration of enzyme production, saccharification, and fermentation into one step. Due to several favorable reasons like single step conversion, no need of adding exogenous enzymes and facilitated product recovery, CBP has gained the attention of researchers worldwide. However, there are several technical and economic barriers which need to be overcome for making consolidated bioprocessing a commercially viable process. Finding a natural candidate CBP organism is critically important and thermophilic anaerobes are preferred microorganisms. The thermophilic anaerobes that can represent CBP mainly belong to genus Clostridium, Caldicellulosiruptor, Thermoanaerobacter, Thermoanaero bacterium, and Geobacillus etc. Amongst them, Clostridium thermocellum has received increased attention as a high utility CBP candidate due to its highest growth rate on crystalline cellulose, the presence of highly efficient cellulosome system and ability to produce ethanol directly from cellulose. Recently with the availability of genetic and molecular tools aiding the metabolic engineering of Clostridium thermocellum have further facilitated the viability of commercial CBP process. With this view, we have specifically screened cellulolytic and xylanolytic thermophilic anaerobic ethanol producing bacteria, from unexplored hot spring/s in India. One of the isolates is a potential CBP organism identified as a new strain of Clostridium thermocellum. This strain has shown superior avicel and xylan degradation under unoptimized conditions compared to reported wild type strains of Clostridium thermocellum and produced more than 50 mM ethanol in 72 hours from 1 % avicel at 60°C. Besides, this strain shows good ethanol tolerance and growth on both hexose and pentose sugars. Hence, with further optimization this new strain could be developed as a potential CBP microbe.

Keywords: Clostridium thermocellum, consolidated bioprocessing, ethanol, thermophilic anaerobes

Procedia PDF Downloads 372